Non-toxic solvent for chromogenic substrate solution and uses thereof

ABSTRACT

The present invention relates to a non-toxic dipolar solvent for chromogenic substrate for detecting presence of lacZ gene and/or gene activity, which comprises a stabilizing amount of a solubilizing agent. The present invention also relates to a method for inducing lac operon in screening assay, comprising the step of contacting an agar plate with at least one essential oil in a concentration sufficient to induce the lac operon. The present invention further relates to a method for detecting the presence of bacteria, comprising the step of contacting an agar plate with at least one essential oil in a concentration sufficient to induce detection of the bacteria.

PRIORITY

This application claims priority to co-pending U.S. application Ser. No.10/533,544, which claims priority to PCT/CA 03/01690 filed Oct. 31,2003, which claims priority to U.S. application Ser. No. 60/422,873filed Nov. 1, 2002 each of which is expressly incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

This invention relates to a non-toxic solvent for preparing chromogenicsubstrate solution and uses thereof.

(b) Description of Prior Art

Many of the cloning and expression vectors in current use (e.g. the pUCseries) carry a short segment of E. coli DNA that contains theregulatory sequences and the coding information for the first 146 aminoacids of the β-galactosidase gene (lacZ). Embedded in this coding regionis a polycloning site that does not disrupt the reading frame butresults in the harmless interpolation of a small number of amino acidsinto the amino-terminal fragment of β-galactosidase. Vectors of thistype are used in host cells that code for the carboxy-terminal portionof β-galactosidase. Although neither the host-encoded nor theplasmid-encoded fragments are themselves active, they can associate toform an enzymatically active protein. This type of complementation, inwhich deletion mutants of the operator-proximal segment of the lacZ geneare complemented by β-galactosidase-negative mutants that have theoperator-proximal region intact, is called α-complementation. The Lac+bacteria that result from α-complementation are easily recognizedbecause they form blue colonies in the presence of the chromogenicsubstrate 5-Bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal)(Horwitz et al. 1964. Substrates for cytochemical demonstration ofenzyme activity. I. Some substituted 3-indoxyl-β-D-galactopyranosides.J. Med. Chem. 7:574.). However, insertion of a fragment of foreign DNAinto the polycloning site of the plasmid almost invariably results inthe production of an amino-terminal fragment that is not capable ofα-complementation. Bacteria carrying recombinant plasmids therefore formwhite colonies. The development of this simple color test has greatlysimplified the identification of recombinants constructed in plasmidvectors of this type. It is easily possible to screen many thousands ofcolonies visually and to recognize colonies that carry putativerecombinant plasmids. The structure of these plasmids is then verifiedby restriction analysis of mini-preparations of plasmid DNA.

To a pre-made LB agar plate containing the appropriate antibiotics, aquantity of a stock solution of X-gal (20 mg/ml in dimethylformamide(DMF) or Dimethyl sulfoxide (DMSO)) and a quantity of a solution ofisopropylthio-β-D-galactoside (IPTG) is added. The stock solution ofX-gal is usually prepared by dissolving X-gal in dimethylformamide orDimethyl sulfoxide which is a toxic solvent presenting also the drawbackof providing solutions that are not stable through time.

IPTG is an important addition to the blue-white screening. The vectorscarrying a segment of DNA derived from the lac operon of E. coli thatcodes for the amino-terminal fragment of β-galactosidase can be inducedby isopropylthio-β-D-galactoside (IPTG). Bacteria exposed to thegratuitous inducer IPTG synthesize both fragments of the enzyme and formblue colonies when plated on media containing the chromogenic substrate5-Bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal).

It would be highly desirable to be provided with new solvents that arenon toxic for preparing chromogenic substrate solutions used inscreening assays, these solvents providing an extended stability of thechromogenic substrate solution.

SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a non-toxicdipolar solvent for chromogenic substrate for detecting presence of lacZgene and/or lacZ gene activity, which comprises a stabilizing amount ofa solubilizing agent.

The solvent in accordance with a preferred embodiment of the presentinvention, wherein the solvent is a microemulsion.

The solvent in accordance with a preferred embodiment of the presentinvention, wherein the solubilizing agent is at least one selected fromthe group consisting of 1-Methylpyrrolidone (NMP), N′-dimethyl propyleneurea (DMPU), Propylene carbonate (PC) and essential oil.

The solvent in accordance with a preferred embodiment of the presentinvention, wherein the essential oil is present in an effectivesolubilizing concentration for dissolving the chromogenic substrate.

The solvent in accordance with a preferred embodiment of the presentinvention, wherein the essential oil is selected from the groupconsisting of Abies alba, Aniba roseodora, Cedrus atlantica, Citrusaurantifolia, Citrus aurantium, Citrus bergamia, Citrus limon, Citrusparadisi, Citrus reticulata, Citrus sinensis, Cupressus sempervirens,Juniperus communis, Juniperus virginiana, Picea mariana, Pinussylvestris, Ravensara aromatica, Rosmarinus officinalis, citrusextracts, pine terpenoids, conifers extracts, limonene oil and linseedoil.

In accordance with the present invention, there is provided acomposition for detecting the presence of lacZ gene and/or lacZ geneactivity comprising the solvent of the present invention and aneffective amount of chromogenic substrate.

In accordance with the present invention, there is provided a method forinducing lac operon in screening assay, comprising the step ofcontacting an agar plate with at least one essential oil in aconcentration sufficient to induce the lac operon.

The method in accordance with a preferred embodiment of the presentinvention, the lac operon being induced in one selected from the groupconsisting of E. Coli, Bacillus subtilis, phage, or in situ tissues.

In accordance with the present invention, there is provided a method fordetecting the presence of bacteria, comprising the step of contacting anagar plate with at least one essential oil in a concentration sufficientto induce detection of the bacteria.

For the purpose of the present invention the following terms are definedbelow.

The term “chromogenic substrate” is intended to mean a substrate thatproduces a color when contacted with an appropriate reagent. Thechromogenic substrate can be one of, but not limited to, X-Gal and IPTG.

All references herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a bacterial culture exhibiting a strong blue colorindicative of lac operon induction without the presence of IPTG whenX-gal dissolved in essential oils;

FIG. 2 illustrates the results of plating a ligation/transformation ontoLB plates containing X-gal dissolved in dimethylformamide (DMF);

FIG. 3 illustrates the results of plating a ligation/transformation ontoLB plates containing X-gal dissolved in NMP and methanol;

FIG. 4 illustrates the results of plating a ligation/transformation ontoLB plates containing X-gal dissolved in NMP, sea pine turpentine andmethanol; and

FIG. 5 illustrates the results of plating a ligation/transformation ontoLB plates containing X-gal solution aged of 16 months;

FIG. 6 illustrates the aging of LB plates at 4° C., room temperature,37° C. and 65° C. (in the clockwise direction); and

FIGS. 7A-7C illustrate LB plates after 2 weeks at various temperatures.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided non-toxicsolvents for dissolving and stabilizing enzyme substrate used inscreening assays.

One enzyme substrate widely used is X-gal, which is a dipolar moleculehaving the formula I:

-   5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside

The X-gal solutions prepared with a non-toxic dipolar solvent of thepresent invention, for example 1-Methylpyrrolidone (NMP), N′-dimethylpropylene urea (DMPU), Propylene carbonate (PC), essential oils or acombination of these, are very stable. In solution at 4° C., the X-galwill keep its activity for more than 6 months. If used to be poured inagar plates containing the proper antibiotic, these plates will remainactive and usable for at least 3 months.

As an example of its non-toxicity, NMP is known as rapidly absorbed andeliminated. It is currently used intravenously in horses as apreanaesthetic. It is also used as an excipient in topicalpharmaceutical formulations in human medicine and in cosmetics.

Essential oil mixes can be used to create a dipolar environment allowingX-gal dissolution. For instance, a combination of citrus extracts, pineterpenoids, limonene and linseed oil was shown to create an environmentallowing the proper dissolution of X-gal.

Non-toxic micro emulsions can also be used to dissolve X-gal. A microemulsion is a thermodynamically stable dispersion of one liquid phaseinto another, stabilized by an interfacial film of surfactant. Thisdispersion may be either oil-in-water or water-in-oil. Micro emulsionsare typically clear solutions, as the droplet diameter is approximately100 nanometers or less. The interfacial tension between the two phasesis extremely low. Emulsions are in contrast unstable, the suspendeddroplets will eventually agglomerate and the dispersed phase will phaseseparate. Emulsion droplet sizes are much larger, typically one micronor more, resulting in a cloudy or milky dispersion. The nature of anemulsion may depend on the mixing of the ingredients and the amount ofenergy put into the mixing process.

A combination of essential oil was extracted with chloroform in order toremove part of the oil phase, and create a semi-precipitated emulsion.For example, 20 ml of citrus extract, pine terpenoids, limonene oils andlinseed oil was mixed with 20 ml of Chloroform by vigorously shaking ina 50 ml falcon tube. The resulting mix was allowed to let stand forapproximately one day, and then the upper phase was transferred to a 15ml falcon tube. The obtained phase is a milky emulsion. It dissolvesX-gal very well. If we let stand the milky phase for approximately oneweek, it will show the development of 5 distinct phases. Isolating eachphase, it was interesting to observe that only phase #2, the milkysub-phase, then still an emulsion, can be used as an active solution todissolve X-gal. The active phase is yellow milky cloudy in appearance.Using 500 μl completely dissolve 0.01 g of X-gal; however, it gives avery opaque and viscous solution in appearance.

Micro emulsions are proper to create dipolar environment allowing acomplete dissolution of X-gal. A mixture of oil, egg yolk or lecithineand acetic acid was sufficient to dissolve X-gal and show blue bacteriawhen used for its function as spread on an agar surface. Linseed oil,which has an average composition of different fatty acids (C16:0palmitic acid 4-9%, C18:0 stearic acid 2-4%, C18:1 oleic acid 14-39%,C18:2 linoleic acid 7-19%, C18:3 linolenic acid 35-66%) is a knowncarrier for lipophilic molecules (as essential oils) and can act as acompound of the oily phase of the emulsion.

A micro emulsion is ideally made of a non-polar liquid mixed with apolar liquid in the presence of a surfactant or amphiphile, which isideally a molecule carrying both polar and non-polar charges. In oursituation, the surfactant, or amphiphilic molecule is X-gal. The role ofthe surfactant is to reduce the interfacial tension between twopartially miscible or immiscible fluids below that obtained when nosurfactant is present.

As shown in Table 1, variation in the composition of the microemulsionhas an impact on the temperature flash point, which is a concern fortransportation purposes.

TABLE 1 X-Gal with Flash point (° C.) 10% water/90% NMP >93 5% water/95%NMP 88 1% seapine/10% water/89% NMP 76 1% seapine/99% NMP 73 10%seapine/90% NMp 47.5

As well, IPTG is a usual and relatively essential addition to thecloning process. Omitting IPTG from the growth medium will decrease theexpression level from plac, blue/white selection is usually not possiblein the absence of IPTG. E. coli lac operon consists of a promoter, atranscriptional regulatory site called the operator (O), a CAP bindingsite (c), and three structural genes (lacZ, lacY and lacA) that aretranscribed as a single polycistronic mRNA. Transcription of the lacoperon is regulated by the lac repressor protein (lad) which is encodedon a gene physically linked to the lac operon. lac operon inducers, suchas IPTG, inactivate the lac repressor protein resulting intranscriptional de-repression of the lac operon. It is possible toartificially induce the lac operon using a nonmetabolizable allolactoseanalogue, isopropylthiogalactoside (IPTG), which binds to the lacrepressor protein.

It is shown here that essential oils are replacing IPTG to induce thelac operon. It was found that compositions comprising essential oils notonly dissolves X-gal, but also enhances the blue color without the needof IPTG. Among different tested essential oils, the most interestingalternative to IPTG is a small amount of the essential oil Sea Pine.Other essential oils extracted from spruce, pine or other conifers arealso candidate to replace IPTG. Using such oil instead of IPTGrepresents different benefits, being non-toxic, all natural, easilybiodegradable, low cost and most importantly, already as a liquidsolution, ready to use.

Monoterpenes and sesquiterpenes were shown to be successful indissolving X-gal and replacing IPTG in screening clones using theblue/white X-gal selection method.

In addition to X-gal, it is also possible to dissolve IPTG with the samesolutions for cloning with bacterial systems needing that stimulation(see Table 2 for examples). These same solutions allow stabledissolution of Ampicillin, tetracycline and Chloramphenicol, providingan approach wherein a complete solution is available to the user. Todissolve Kanamycin, a solution with a base of micelles in aqueoussolution is essential.

TABLE 2 Strain (lacZ)M15 lacIq F′ Cam R Kan R Str R Tet R CommentsDH5-alpha x Invitrogen: (lacZYA)U169 DH5-alpha E x Invitrogen;(lacZYA)U169 DH5-alpha F′ x x Invitrogen; (lacZYA)U169 DH5-alpha xInvitrogen; T1R (lacZYA)U169 DH10B x Invitrogen DH10Bac x Invitrogen;for producing recombinant baculovirus molecules DH10B T1 R x InvitrogenGeneHogs x Invitrogen; D10B derivative INV-alpha F′ x x Invitrogen,(lacZYA)U169 JM83 x x ATCC 35607 Select 96 x Promega TB1 x x NEB; comeswith pMAL system TOP10 x Invitrogen TOP10/P3 x x x x Invitrogen; AMP RABLE C x x x x x Stratagene; lower plasmid copy number ABLE K x x x x xstratagene; lower plasmid copy number AD494 BB4 x x x x =LE392.23 BMH71-18 x x x x mutS DH5-alpha F′ x x x x Invitrogen IQ DH-alpha FT x x xx Invitrogen; (lacZYA)U169 DH11S x x x Invitrogen; mainly for ssDNAproduction with phagemid/m13 DH12S x x x x Invitrogen Electro Ten x x xx x Stratagene blue ER1727 x x x x x NEB ER2267 x x x NEB ER2738 x x xNEB INV110 x x x x Invitrogen; to produce umethylated DNA JM101 x x xATCC33876 JM103 x x x x ATCC 39403 JM105 x x x x ATCC47016 JM107 x x xATCC47014 JM109 x x x ATCC 53323; to maintain F′ grow on M9 with 1 mMthiamine JM110 x x x x ATCC 47013 NM522 x x x ATCC 47000 NM527 SCS110 xx x x Stratagene Solo Pack x x x x x Stratagene gold SOLR x x x xStratagene; excision assist strain Stbl4 x x x x Invitrogen; to cloneunstable DNA SURE x x x x x Stratagene SURE-2 x x x x x x Stratagene TG1x x x TKX1 x x x x x Stratagene; for prot production TOP10 F′ x x x x xW3110 XL1-Blue x x x x Stratagene XL1-Blue x x x x Stratagene MRF′XL1-Blue x x x x x Stratagene MRF′ Kan XL2-Blue x x x x x StratageneXL2-Blue x x x x x Stratagene MRF′ XL10gold x x x x x StratageneXL10gold x x x x x Stratagene Kan R XL mutS Kan S x x x x Stratagene XLmutS Kan R x x x x x stratagene XLOLR x x x x Stratagene; excisionassist strain XPORT x x x G1698 x C1724 x 96 pack gold x x x x x EC100 xEpicentre; pIndigo EC300 x Epicentre; copy control BAC CC300 xEpicentre; copy control BAC EPI100 ? ? Epicentre; fosmid EPI300 ? ?Epicentre; fosmid AG1 DH1 derivative BL21 (DE3) x for prot expression;some also Cam R or Kan R BNN93 C600 ATCC 23724 CJ236 x x DB3.1Invitrogen; for propagating vector with ccdB gene DH1 ATCC 33849 DH5alpha Invitrogen; MCR (lacZYA)U169

In a preferred embodiment of the present invention, individual LB platesare prepared adding 100 μl of X-gal solution to the surface of a LBplate being at room temperature and spreading evenly across the surface.The plate is dried before use. X-gal containing LB+Amp plates are stablefor up to 90 days when stored at 4° C.

Batches can be prepared by aseptically adding X-gal solution directly tomelted LB agar (temperature 50° C.). 100 μl of the solution should beused for every Petri dish. For example, for each 500 ml add 2000 μl ofX-gal solution. Mix well (for 3 to 5 minutes) and pour as you normallywould. Let cool. X-gal LB plates are stable for at least 3 months whenstored at 4° C. X-gal can be added before or after the addition ofselective antibiotics to the medium. For best mixing results, a magneticstir bar should be added during the autoclaving process or a sterilemagnetic stir bar could be ascetically added after autoclaving.

In a preferred embodiment of the present invention, X-gal solution isprepared as follow:

NMP or DMPU Only

Add 7 ml of either NMP or DMPU to 1 gram of X-gal. Stir until dissolvedand bring to 10 ml with NMP or DMPU (whichever is already used).

NMP or DMPU with Essential Oil

Add 3.5 ml of either NMP or DMPU to 1 gram of X-gal. Stir untildissolved and bring to 5 ml with NMP or DMPU (whichever is alreadyused). Mix with 5 ml of essential oil until thoroughly mixed.

NMP/Methanol Solution

Add 7 ml of either NMP or DMPU to 1 gram of X-gal and stir untildissolved. Bring to 10 ml with NMP or DMPU (whichever is already used).Add 90 ml of methanol and mix thoroughly.

Example 1 Essential Oils as Solvent and IPTG Replacement

Using 0.01 g X-gal in 500 μl of TURPENOID NATURAL® (comprising acombination of citrus extracts, pine terpenoids, limonene and linseedoil) provides excellent dissolution of X-gal and without affectingbacterial growth. It also provides a strong blue color without the needof IPTG, as shown in FIG. 1.

Example 2 Dissolution of X-gal

10 mg of X-gal powder were successfully dissolved in the solventsdescribed in the Table 3 below.

TABLE 3 Mix T1− Citrus lemon/Citrus 700 μl sinensis Pinus pinaster 300μl Mix T2− Cupressus sempervirens  50 μl Pinene (Sigma)  50 μl Campheroil 100 μl Citrus sinensis 800 μl Mix T3− NMP 100 μl Methanol (only to900 μl increase total volume): % NMP: 10% Mix T4− PC 200 μl Methanol(only to 400 μl increase total volume): % PC: 33%

Example 3 Dissolution in NPM and NMP/Methanol

A 10× X-gal/NMP solution could be stored at ⁻20° C. without freezing.Over time, the solution will take a very pale yellow color that does notseem to darken over time. A 10× X-gal/NMP solution is easily diluted to1× in anhydrous methanol or 95% ethanol. When diluted in methanol orethanol, the solution will not freeze.

When the 1× X-gal/NMP/methanol is stored for 16 weeks at 4° C., there isno loss in X-gal activity as measured by applying 100 μl to a LB plate,streaking E. coli pUC19 transfectant, incubation at 37° C. andexamination for blue colonies after 16 hours.

Tables 4 and 5 are showing the results obtained by testing the activityof X-gal in solution with different solvent on a weekly basis. In Table4, the tested solutions were stored at 4° C. At weekly intervals, 100 μlwas applied to an LB+Amp plate and streaked with E. coli containingpUC19. The plate was incubated overnight at 37° C. The following morningthe plate was examined for blue colonies. In Table 5, LB+Amp plates wereprepared and stored at 4° C. At weekly intervals, a plate was removedand streaked with E. coli containing pUC19. The plate was incubated at37° C. overnight. The blueness of the colonies was then scored.

TABLE 4 Weekly Testing of X-gal/NMP/Methanol solution Week Test Results0 + 1 + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12 + 13 + 14 + 15 +16 + 17 + 18 + 19 + 20 +

TABLE 5 Weekly testing of X-gal/NMP or X-gal/NMP/oil Date NMP plateOil + NMP plate 0 + + 1 + + 2 + + 3 + + 4 + + 5 + + 6 + + 7 + + 8 + +9 + + 10 + + 11 + + 12 + + 13 + + 14 + +

Example 4 Dissolution with Essential Oils

It was shown that when X-gal was first dissolved in NMP, mixed one toone with essential oil, and then diluted in methanol to 10 mg/ml themixture would ultimately yield colonies that are darker blue than whenX-gal is made up in dimethylformamide.

This work was repeated and expanded to include other essential oils. Theoils tested were: Natural Orange Terpene Solvent (Eco-House); Blue GumEucalyptus Organic (Divine Essense); Atlas cedarwood (Pranarom); SeaPine Turpentine (Pranarom) and Natural Turpenoid.

In this experiment, a 100 mg/ml solution of X-gal in NMP was prepared. Aone to one mix with each essential oil was then made using thissolution. The net X-gal concentration is now 50 mg/ml. This solution wasthen dilute 5× with 100% methanol such that the final concentration ofX-gal is 10 mg/ml. One hundred microliters of this solution is thenapplied per LB plate.

As a control, 50 μl of a 20 mg/ml solution of X-gal dissolved indimethylformamide was applied to one LB plate.

E. coli containing pUC19 was streaked onto each “X-gal spread” LB platefor isolated colonies. The plates were incubated at 37° C. overnight.After incubation, the growth on each X-gal plate was scored forblueness. The ranking was as follows (least blue to most blue): A(least); B and X-gal/DMF; C, D and E (most blue).

The results showed that when either Atlas Cedarwood, Sea Pine Turpenineor Natural Turpenoid were mixed with X-gal/NMP/methanol there was anenhanced blueness of the E. Coli pUC containing bacteria.

During assays with Sea Pine Turpentine, the amount of Sea PineTurpentine added to the X-gal/NMP solution was serially diluted one intwo four times, mixed with methanol and then spread onto LB plates. Theamount of X-gal added to each plate was the same. The plates were thenstreaked with E coli containing pUC19 and incubated overnight.

An examination of the plates showed that the bluest colonies were thoseobtained with the original amount of Sea Pine Turpentine gave thedeepest blue color.

In the previous experiments, the final X-gal concentration was 10 mg/ml.The concentration of X-gal was reduced to 7.5, 5 and 2.5 mg/ml. Plateswere prepared and streaked. The net result was that there was asignificant drop in blue coloration when the X-gal was dropped from 7.5to 2.5 mg/ml. In a preferred embodiment of the present invention, X-galis in a concentration of 10 mg/ml.

Ligation assays were performed using lambda DNA digested with PstI andpUC19 digested with PstI and CTAP treated. The completed ligation wastransformed into DH5α and plated onto LB plates containing:

(a) X-gal dissolved in Dimethylformamide

(b) X-gal dissolved in NMP and methanol

(c) X-gal dissolved in NMP, Sea Pine Turpentine and methanol

The plates were incubated overnight. The results for a, b and c areshown in FIGS. 2, 3 and 4, respectively. For all three plating, therewere both white and blue colonies. Moreover, it is shown that the bluescolonies of FIG. 4 are of a more intense blue than the ones of FIGS. 2and 3.

It is possible to incorporate X-gal directly in molten LB agar. To testthis with the X-gal solution of the present invention, 500 ml of LB agarwas made, autoclaved and cooled to about 50° C. To this 2 ml of a 10mg/ml X-gal/NMP/SeaPine/methanol solution was added and mixed. Upon theaddition of the X-gal solution, there was a cloudy appearance throughoutthe agar as it mixed. Mixing for 3 to 4 minutes did not disperse thecloudiness. However, when the plates were poured, the cloudinessdispersed upon cooling and solidification. A slight surface cloudinesswas noted several hours later when the plates were inverted forincubation overnight at room temperature. However, the next day theplates looked normal.

One plate was used to streak a white and a blue colony and incubatedovernight at 37° C.

Two plates were left at 25° C. These two plates served as shippingsimulators. The plates were tested after 5 days and were streaked with ablue and white colony and incubated overnight.

Aging studies have been set up for the liquid product both with andwithout Sea Pine Turpentine. The studies conducted with theX-gal/NMP/methanol solution shown that the product is stable for 17weeks at 4° C. with only a minor pale yellow color developing over time.This has being conducted at the same time with theX-gal/NMP/SeaPine/methanol product.

Example 5 Aging Studies

X-gal solution using NMP and/or essential oil were shown to have animprove shelf-life. Table 6 is providing life span of X-gal solutions.

TABLE 6 Temperature Time  4° C. 16 months Room temperature  4 months 37°C. 1 month 65° C. 1 month

Moreover, it had been found that XGaI Petri dishes were still activewhen conserved at 4° C. during as long as 12 months.

Method and Results

Longest-term recipe is 1 Jun. 2002 in amber bottles and stored at 4° C.The recipe is 10% NMP and 90% methanol. The solution was last test Sep.25, 2003 and found to be functioning fine (see FIG. 5). Currently, onlya pale yellow color and not crystals.

X-gal containing LB plates were prepared Jul. 30, 2002. Two sets ofplates were poured and stored at 4° C. only. The first set was X-galdissolved in NMP then an equal volume of natural turpenoid extract wasadded followed by ethanol. The final X-gal concentration in the solutionwas 10 mg/ml. Ampicillin prepared in water was added separately to themolten agar before pouring.

Each week one plate was removed, streaked with pUC/DH5α and DH5α andincubated at 37° C. overnight. During the course of the experiment, thestreaked pUC/DH5α always turned blue and the DH5α did not grow. Up untilJul. 30, 2003, both Ampicillin and X-gal were functioning.

Tables 7-9 provide results from more aging studies performed withsolutions in accordance with the present invention

TABLE 7 10% NMP Main Ingredients May 12 June 3 June 9 June 13 June 27July 8 Aug 5 Aug 15 Sept 22 X-gal (ethanol) No crystals, Clear, noClear, no Yellow, no Yellow, no Yellow, no Yellow Slightly Slightly May5, 2003 clear crystals crystals crystals crystals color, no crystals,yellow, no yellow, no solution, ok ok crystals ok crystals ok crystalsok Ok X-gal No crystals, Clear, no Clear, no Clear, no Clear, no Clear,no Clear, no Clear, no Clear, no (methanol) clear crystals crystals okcrystals ok crystals ok crystals crystals crystals crystals May 1, 2003solution ok ok Ok Ampicillin + No crystals, Yellow, yellow yellow color,yellow, no Yellow, no Yellow, no Yellow, no Yellow, no X-gal slightyellow No crystals, color, no no crystals crystals Ok crystals crystals,crystals crystals May 6, 2003 color Ok ok crystals Ok ok ok Ok ok Ok okOk ok Ok ok Ok ok Amp in NMP + NA Pale yellow Yellow, no Yellow, noYellow, no Yellow, no Yellow, no Yellow, no Yellow, no X-gal Ok okcrystals crystals crystals crystals crystals crystals crystals Ok Ok May15, 2003 Ok, ok Ok, ok Ok, ok Ok ok Ok ok Chloramphenic No crystals,Pale yellow, Pale yellow, Pale yellow, Pale yellow, Pale yellow, Paleyellow, Pale yellow, Pale yellow, no of IPTG (low) + clear no crystals,no crystals no crystals no crystals no crystals no crystals no crystalscrystals X-gal solution Ok, ok Ok ok Ok ok Ok Ok Ok ok Ok ok Ok ok May5, 2003 Ok, ok Chloramphenicol No crystals, Pale yellow, Pale yellowPale yellow, yellow Pale yellow, Pale yellow, Pale yellow, Pale yellow,no IPTG (high) + clear no crystals, no crystals no crystals color, no nocrystals, no crystals, no crystals crystals Ok Ok X-gal solution Ok, okOk ok Ok ok crystals Ok ok, ok ok ok May 5, 2003 Ok, ok okTetracycline* + Yellow, no Yellow, Darker Darker Darker Dark yellow,Dark yellow, Yellow, no Yellow, no X-gal crystals, clear, no yellow, noyellow, no yellow no crystals no crystals crystals, ok crystals, ok ok(Apr. 4, 2003) No growth crystals, crystals crystals color, no Ok ok Okok ok of DH5α No growth Ok ok crystals of DH5α ok ok TetracyclineYellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, noYellow, no Yellow, no Yellow, no (low) + X-gal crystals crystalscrystals crystals crystals crystals crystals, crystals crystals Ok Ok(May 5, 2003) Ok ok Ok, ok Tetracycline Yellow, no Yellow, no Yellow, noYellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no(high) + X-gal crystals crystals crystals crystals crystals crystalscrystals, crystals crystals (May 5, 2003) Ok, ok Ok, ok Ok Ok

TABLE 8 AGING TEST #1 Aging test Aging test Formulations Start date (#weeks) Result (# weeks) DMF + X-Gal RT 17 Sep. 2003 1 week Appearance ofsoln: clear, Intensity 4+ 4 weeks −20 mid-july-03 ~9 weeks Appearance ofsoln: clear, Intensity 4+ ~12 weeks BT lot no. 024 (25% NMP) 65 C.  17Sep. 2003 1 week Appearance of soln: yellow, Intensity 4+ 4 weeks 37 C. 17 Sep. 2003 1 week Appearance of soln: light yellow, Intensity 4+ 4weeks RT 21 Aug. 2003 5 weeks Appearance of soln: clear, Intensity 4+ 8weeks 4 C. 5 Aug. 2003 ~7 weeeks Appearance of soln: clear, Intensity 4+~10 weeks BT lot no. 004 RT 21 Aug. 2003 5 weeks Appearance of soln:light yellow, Intensity 4+ 8 weeks 4 C. 18 Feb. 2003 ~31 weeksAppearance of soln: light yellow, Intensity 4+ ~34 weeks BT Amp, lot 019RT 21 Aug. 2003 5 weeks Appearance of soln: yellow, Intensity 3+ 8 weeks4 C. 16 May 2003 ~18 weeks Appearance of soln: yellow, Intensity 3+ ~21weeks All were tested with DH5 + TrueBlue BT lot no. 024 (25% NMP) hasan intensity rated higher than others probably because the resultingevaporation made it more [ ]

TABLE 9 AGING TEST NEW FORMULATIONS Formulations Start date Week 2Result W4 (M1) A + X-Gal/IPTG 65 C. 28 Sep. 2003 14 Oct. 2003 Appearanceof soln: VERYyellow, Intensity 3+ 28 Oct. 2003 37 C. 28 Sep. 2003 14Oct. 2003 Appearance of soln: light yellow, Intensity 4+ 28 Oct. 2003 RT28 Sep. 2003 28 Oct. 2003  4 C. 28 Sep. 2003 14 Oct. 2003 Appearance ofsoln: clear, Intensity 4+ 28 Oct. 2003 −20 28 Sep. 2003 28 Oct. 2003 D +X-Gal/IPTG 65 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln:VERYyellow, Intensity 3+ 28 Oct. 2003 37 C. 28 Sep. 2003 14 Oct. 2003Appearance of soln: light yellow, Intensity 4+ 28 Oct. 2003 RT 28 Sep.2003  4 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: clear,Intensity 4+ −20 28 Sep. 2003 28 Oct. 2003 K + X-Gal/IPTG 65 C. 28 Sep.2003 14 Oct. 2003 Appearance of soln: brown-orange, Intensity 3+ 28 Oct.2003 37 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: yellow,Intensity 4+ 28 Oct. 2003 RT 28 Sep. 2003 28 Oct. 2003  4 C. 28 Sep.2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct. 2003−20 28 Sep. 2003 28 Oct. 2003 BT PLUS 65 C. 28 Sep. 2003 14 Oct. 2003Appearance of soln: greenish-brown, Intensity 3+ 28 Oct. 2003 lot no.028, 25% NMP 37 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: lightyellow, Intensity 4+ 28 Oct. 2003 RT 28 Sep. 2003 28 Oct. 2003  4 C. 28Sep. 2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct.2003 −20 28 Sep. 2003 28 Oct. 2003 DMF + X-Gal/IPTG 65 C. 28 Sep. 200314 Oct. 2003 Appearance of soln: orange, Intensity −−− 37 C. 28 Sep.2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct. 2003RT 28 Sep. 2003 28 Oct. 2003  4 C. 28 Sep. 2003 28 Oct. 2003 −20 28 Sep.2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct. 2003DMSO + X-Gal/IPTG 65 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln:greenish-brown, Intensity −−− 37 C. 28 Sep. 2003 14 Oct. 2003 Appearanceof soln: light yellow, Intensity 4+ 28 Oct. 2003 RT 28 Sep. 2003 28 Oct.2003  4 C. 28 Sep. 2003 28 Oct. 2003 −20 28 Sep. 2003 14 Oct. 2003Appearance of soln: clear, Intensity 4+ 28 Oct. 2003 Test 14 oct wasdone with DH5 + TrueBlue Test 28 oct was done with XL1 + PUC (alsosolutions are 65 C. were rought back to 1400 uL)

In FIG. 6, dishes from the BT lot no. 024, as described in Table 8 areillustrated. The two top left dish was conserved at 4° C., the top rightdish was conserved at room temperature, the bottom left dish wasconserved at 37° C. and the bottom right dish was conserved at 65° C.

In FIG. 7A, dishes from the lot A, as described in Table 9, areillustrated. The top dish was conserved two weeks at 4° C., the bottomleft dish was conserved two weeks at 37° C. and the bottom right dishwas conserved two weeks at 65° C.

In FIG. 7B, dishes from the lot 028, as described in Table 9, areillustrated. The top dish was conserved two weeks at 4° C., the bottomleft dish was conserved two weeks at 37° C. and the bottom right dishwas conserved two weeks at 65° C.

In FIG. 7C, dishes from the lot DMSO, as described in Table 9, areillustrated. The top dish was conserved two weeks at −20° C., the bottomleft dish was conserved two weeks at 37° C. and the bottom right dishwas conserved two weeks at 65° C.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

The invention claimed is:
 1. A biochemical solvent for a chromogenicsubstrate for detecting presence, activity, expression, and/ornon-expression of a lacZ gene, the solvent comprising a compoundselected from the group consisting of 1-methylpyrrolidone (NMP),N¹-dimethyl propylene urea (DMPU), propylene carbonate (PC), andcombinations thereof, and a lipophilic essential oil creating a dipolarenvironment, in a concentration sufficient to induce a lac operon,selected from the group consisting of Abies alba, Aniba roseodora,Cedrus atlantica, Citrus aurantifolia, Citrus aurantium, Citrusbergamia, Citrus limon, Citrus paradisi, Citrus reticulata, Citrussinensis, Cupressus sempervirens, Juniperus communis, Juniperusvirginiana, Picea mariana, Pinus pinaster, Pinus sylvestris, Ravensaraaromatica, Rosmarinus officinalis, citrus extracts, pine terpenoids,conifers extracts, limonene oil, linseed oil, and combinations thereof.2. The solvent of claim 1 where the compound is at a concentrationsufficient to maintain substrate activity for more than six months insolution at 4° C.
 3. The solvent of claim 1 being a microemulsion. 4.The solvent of claim 1 as a ready-to-use non-toxic liquid.
 5. Acomposition for detecting the presence of lacZ gene comprising thesolvent of claim 1 and an effective amount of a chromogenic substrateselected from the group consisting of substitutedindolyl-β-D-galactoside, halo-substituted indolyl-β-D-galactoside,5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal), andcombinations thereof.
 6. A composition for detecting the presence oflacZ gene comprising the solvent of claim 1, and an effective amount ofsubstituted indolyl-β-D-galactoside or halo-substitutedindolyl-β-D-galactoside chromogenic substrate selected from the groupconsisting of 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside,6-chloro-3-indolyl-β-D-galactopyranoside,n-methylindolyl-β-D-galactopyranoside, and5-iodo-3-indolyl-β-D-galactopyranoside.
 7. A composition for detectingpresence, activity, and/or expression of a lacZ gene, the compositioncomprising a chromogenic substrate to visibly indicate presence,activity, and/or expression of the lacZ gene, the substrate selectedfrom the group consisting of substituted indolyl-β-D-galactoside orhalo-substituted indolyl-β-D-galactoside; a biochemical solventcomprising at least one solubilizing agent selected from the groupconsisting of 1-methylpyrrolidone (NMP), N¹-dimethyl propylene urea(DMPU), propylene carbonate (PC), and combinations thereof, and at leastone essential oil creating a dipolar environment, wherein the at leastone essential oil is at a concentration sufficient to induce a lacoperon.
 8. The composition of claim 7 where the inducer is notisopropylthio-β-D-galactoside (IPTG).
 9. The composition of 7 furthercomprising at least one antibiotic with the substrate.
 10. Thecomposition of claim 9 where the antibiotic is selected from the groupconsisting of ampicillin, tetracycline, chloramphenicol, kanamycin, andcombinations thereof.
 11. A self-contained pre-cast medium for detectingactivity and/or expression or non-expression of a lacZ gene, the mediumcomprising an agar matrix and in or on the agar matrix a chromogenicsubstrate selected from the group consisting of substitutedindolyl-β-D-galactoside, halo-substituted indolyl-β-D-galactoside, and5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal); a biochemicalsolvent comprising at least one solubilizing agent selected from thegroup consisting of 1-methylpyrrolidone (NMP), N¹-dimethyl propyleneurea (DMPU), propylene carbonate (PC), and combinations thereof, and atleast one lipophilic essential oil creating a dipolar environment,wherein the essential oil is at a concentration sufficient to induce alac operon.
 12. The medium of 11 further comprising at least oneantibiotic with the substrate.
 13. The medium of claim 12 where theantibiotic is selected from the group consisting of ampicillin,tetracycline, chloramphenicol, kanamycin, and combinations thereof. 14.The medium of claim 11 having functional stability for at least threemonths 4° C.
 15. The medium of claim 11 in a ready-to-use plate.
 16. Acomposition for detecting presence, activity, expression, and/ornon-expression of a lacZ gene, the composition comprising a chromogenicsubstrate providing a visible indication of the presence, activity,expression, and/or non-expression of the lacZ gene, a non-toxicmicroemulsion biochemical solvent selected from the group consisting of1-methylpyrrolidone (NMP), N¹-dimethyl propylene urea (DMPU), propylenecarbonate (PC), and combinations thereof facilitating substratedissolution, and at least one lipophilic essential oil creating adipolar environment and replacing isopropylthio-β-D-galactoside (IPTG)at a concentration sufficient to induce a lac operon.
 17. Thecomposition of claim 16 as a ready-to-use liquid.
 18. A composition fordetecting presence, activity, expression, and/or non-expression of alacZ gene, the composition comprising a chromogenic substrate providinga visible indication of the presence of lacZ gene and/or lacZ geneactivity; a first phase comprising at least one solubilizing agentselected from the group consisting of 1-methylpyrrolidone (NMP),N¹-dimethyl propylene urea (DMPU), propylene carbonate (PC), anessential oil, and combinations thereof; a second phase comprising seapine turpentine in a concentration of at most 1% v/v of the compositionsufficient to induce a lac operon, and water; the composition being amicroemulsion facilitating substrate dissolution and having a flashpoint greater than 73° C.
 19. The composition of claim 18 where theflash point is 76° C.
 20. A composition comprising a chromogenicsubstrate selected from the group consisting of substitutedindolyl-β-D-galactoside, halo-substituted indolyl-β-D-galactoside, or5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal); a1-methylpyrrolidone (NMP) solubilizing agent; and sea pine turpentine ina concentration sufficient to induce a lac operon; the compositionhaving a flash point greater than 73° C.
 21. The composition of claim 20where the substituted indolyl-β-D-galactoside or halo-substitutedindolyl-β-D-galactoside is selected from the group consisting of5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside,6-chloro-3-indolyl-β-D-galactopyranoside,n-methylindolyl-β-D-galactopyranoside, and5-iodo-3-indolyl-β-D-galactopyranoside.
 22. The composition of claim 20having about 1% sea pine turpentine.
 23. The composition of claim 20having a flash point of 76° C.
 24. The composition of claim 20 havingabout 89% NMP.
 25. The composition of claim 20 being transport safe. 26.The composition of claim 20 lacking methanol.